الصرفة والتطبیقیة 2009) 4(2 2 المجلد مجلة ابن الھیثم للعلوم تطویر طریقتین طیفیتین لتقدیر االمتربتلین في المستحضرات الصیدالنیة باالعتماد على دراسة المحددات أحادیا وبطریقة السمبلكس للوصول إلى الظروف الفضلى عالء كریم محمد ة التربیة ابن الهیثم،جامعة بغدادقسم الكیمیاء،كلی الخالصة ه النقـي وفـي األقـراصان وبـسیطتان سـهلتان طیفیتـا طریقتت اقترح تقـدیر االمتـربتلین بـشكل ى .ن ل تعتمـد الطریقـة األولـى علـ بـ قــال شـحنة بــین االمتـربتلین واه ین اتكـوین معقـد انت ـیانواثل تتراسـ يمثـل لاللكترونــات مـع ستقبل بــا ــا.مـ تفاعـل أقــصى اظهـر ن تج ال ة . 9 نــانومیتر فـي مـذیب االسـیتونتریل عنـد دالــة حمـضیة مقـدارها 470امتـصاص عنـد طـول مـوجي مقـداره وفـي الطریقـة الثانیــ طـول مـوجي تقیـس زول األخـضر عنـد لبرومـوكری ة مـع ا راسـ ألیـوني المتكـون مـن تفاعـل الـدواء قیـد الد امتـصاصیة معقـد االزدواج ا 3. 5 دالة حمضیة مقدارها نانومیتر عند415مقداره فـي دراسـة المتغیـرات للوصـول إلـى الظـروف ا طریقتتطبق ا التقلیدیـة وطریقـة الـسمبلكس المحـورة تغیر المحددات أحادیـ اد امج الـسمبلكس الهندســي ثالثـي اإلبعـ ـانون أظهـرت النتـائج التــي تـم الحـصول علیهـ.الفـضلى للتفـاعلین باسـتخدام برنـ ة لقـ ا مطاوعـ ـایكرو غـرا8 -100 و 6-70 بییـر فـي مـدى تركیـز یـتراوح مـن مـل.م مـ - 1 مقــدارها یم معامـل امتـصاص مـوالي و 2275 وبقـ یانواثیلیین وبرومـوكریزول األخـضر علـى التـوالي1-سـم .1-مـول . لتـر1475 ت قـیم حـدود الكـشف مـساویة إلـى . لطریقتـي تتراسـ كانـ لم.م مایكرو غرا0.034 و0.043 -1 سـم.م مـایكرو غـرا0.188 و 0.122 وقـیم حـساسیة سـاندل هـي -1 . للطـریقتین علـى التـوالي . ن بنجاح لتقدیر االمتربتلین في أقراص دوائیة من مناشى مختلفة وكانت دقة وتوافق النتائج مقبولةاطبقت الطریقت IBN AL- HAITHAM J. FO R PURE & APPL. SCI. VO L. 22 (4) 2009 Development of Two New Spectrophotometeric Methods for the Determination of Amitriptyline in Pharmaceutical Preparation Using Univariate and Simplex Optimization A. k . Mohammed Department of Chemistry, College of Education,Ibn Al-Haitham University of Baghdad Abstract Two simple and sensitive spectrophotometric methods are proposed for the determination of amitriptyline in its pure form and in tablets. The first method is based on the formation of charge- transfer complex between amitriptyline as n-donor and tetracyano-ethy lene (TCNE) as π- acceptor. The product exhibit absorbance maximum at 470 nm in acetonitrile solvent (pH =9.0 ) . In the second method the absorbance of the ion- pair complex, which is formed between the soughted drug and bromocresol green (BCG), was measured at 415 nm at ( pH=3.5) . In addition to classical univariate optimization, modified simplex method (M SM ) was app lied in the optimization of the variable affecting the color p roducing reaction by a geometric simplex in three dimensions of space. Beers , law was obeyed in the concentration ranges 6.0-70 and 8.0-100 µg.ml -1 with molar absorbitivites of 2275 and 1475 l. mol -1 cm -1 for TCNE and BCG methods resp ectively. The limits of detection of the two methods are 0.043 and 0.034 µg.ml -1 and their Sandells sensitivity values are 0.122 and 0.188 µg.ml-1 resp ectively. Introduction Amitriptyline is chemically 3-(10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5-y lidene)- N,N-dimethyl-1-propanamine, a tricyclic antidepressant drug, widely used for treating clinical depression, neuropathic pain , nocturnal enuresis, and attention-deficit hyp eractivity disorder (ADHD) [1] , but it has also been used successfully for headache, anxiety , smoking cessation, bulimia nervosa, persistent hiccups and as an adjunct in schizophrenia [2]. The vital importance of this drug prompted the development of various analytical methods for its determination , these methods include capillary electrophoresis [3,4] high performance liquid chromatography [5,6], gas chromatography [7,8], potentiometric [9,10] , chemometric [11], and spectrophotometry [12-18]. π-acceptors such as 2,3-dichloro-5,6-dicyano-p -benzo-quinone(DDQ), 7,7,8,8- tetracyanoquino-dimethane(TCNQ), tetracyano-ethylene(TCNE), 2,4,7-trinitroflurene-9- one(TNF), 2,5-dihydroxy-3,6-dichloro-p -benzoquinone( p- chloranilic acid) are known to yield charge transfer complexes and radical ions with a variety of electron donors such as cephalosporins [19] , gabapentin [20] , loratadine [21] and ranitidine [22] . On the other hand, ion- pair extraction sp ectrophotometry has been received aconsiderable attention for quantitative estimation of pharmaceutical compound . Bromophenol blue (BPB), methylene blue (M B) , bromocresol purp le (BCP) and thymol blue (TB) were widely used as ion- pairing reagents for the quantitative analysis of many pharmaceutical compounds [23-25] . IBN AL- HAITHAM J. FO R PURE & APPL. SCI. VO L. 22 (4) 2009 In experimental chemistry, the optimization of technical systems is the process of adjust ing the control variables to find the levels that achieve the best opt imization. Usually , many conflicting response must be optimized simultaneously. In lack of systematic approaches the optimization is done by “ trial-and- error”, or by changing one control variable at a time while holding the rest constant, such methods requires a lot of experiments to be carried out. Simplex optimization of experimental parameters was first introduced by Spendley [26] and then modified by Nelder [27] and Aberg [28] . The method found a lot of applications in the felid of analytical chemistry [29-32] , because it offers the capability of optimizing several factors simultaneously depending on a statistical design search to find the maxima or minima of response, by rejecting the point producing the worst response and a replacement of it by the new point which is obtained statistically. The present work established an improved spectrophotometric method for the determination of amitriptyline by exploiting its basic nature and electron- donating p roperty. The determination is based on charge- transfer reaction with TCNE and ion – association complexation with Bromocresol green (BCG) and the optimization of chemical sp ectrophotometric variables of the proposed methods namely pH, reagent amount and reaction time were studied by using both classical univariate and modified simplex. The multivariate simplex op timization was carr ied out via computer program [33]adapted to fit personal micro- computer. Experime ntal Apparatus: A Shimadzu ( model 1601 UV- visible sp ectrophotometer from Shimadzu, Koyoto, Japan ) with 1cm glass cells was used for absorbance measurements.pH – meter model PW-9421 from Philips was used for all pH measurements . Materials and Reagents All chemical used were of analytical reagent grade unless otherwise- amitriptyline- hydrochloride standard powder materials (purity 99.8%) were provided from the State Company for Drug Industries and M edical Appliances Samara-Iraq (SDI). 1- TCNE 1.6x 10 -3 M solution, p repared by dissolving 20.5 mg of the reagent in 100 ml of acetonitrile by using volumetric flask. 2- BCG 1x10 -3 M solution p repared by dissolving 36.0 mg of the reagent in 50 ml dist illed water by using volumetric flask. 3- Phthalate buffer (p H=3.60).To 250 ml of 0.2M potassium hydrogen phthalate 11.90 ml of 0.2M HCl was added and then the solution was diluted to a final volume 1000ml with dist illed water [34] . Standard amitriptyline solutions: 1- Solution for TCNE procedure (250 µg.ml 1- ): 50 mg of amitriptyline base was dissolved in 50 ml of methanol, then the solution was made alkaline (pH=9.0) with a dropwise of 0.2N sodium hydroxide.The solution was quantitatively transferred into a separating funnel and shaken with four 10 ml portions of chloroform.The extracts were pooled by filtration through a filter paper containing anhydrous sodium sulphate into a 200 ml volumetric f lask and diluted to volume with chloroform. This was diluted to get a working concentration of 100 µg/ml . IBN AL- HAITHAM J. FO R PURE & APPL. SCI. VO L. 22 (4) 2009 2- Solution for BCG procedure (250 µg.ml -1 ) : 50 mg of amitrip tyline base was dissolved in 200 ml of methanol by using vo lumetric flask. Procedures: Calibration graphs 1- TCNE method: Serial volumes of standard solution ranging fro m 0.60 to 7.0 ml were transferred to 10 ml volumetric flasks , then 1.50 ml of TCNE reagent was added, and allowed to stand for 30 min at 25 0 C and then diluted to volume with acetonitrile. The absorbance was measured at 470nm against reagent blank. 2- BCG method: Serial volumes of standard solution ranging 0.32 to 4.0 ml were transferred individually into 25 ml separating funnel, then 1 ml of BCG solution and three ml phthalate buffer (pH= 3.6 ) were added.The formed complex was extracted for 2 min with two 5 ml portions of chloroform. The extracts were pooled by filtration through a filter paper containing anhy drous sodium sulphate into a 10 ml volumetric flask and diluted to volume with chlorofor m, and then the absorbance was measured at 415 nm against reagent blank. Procedure for the determination of amitriptyline in pharmaceutical preparation: TCNE method: Ten tablets were finely powdered and mixed .An accurately weighed quantity equivalent to the drug base concentration mentioned in the standard solution preparation was dissolved by shaking with 50 ml distilled water . The solution was made alkaline (pH=9.0) with a drop wise addition of 0.2N sodium hydroxide. The resulted solution was quantitatively transferred into a separating funnel and shaken with four 10 ml portions of chloroform. The extracts were pooled by filtrat ion through a filter paper containing anhydrous sodium sulphate into a 200 ml volumetric f lask and diluted to volume with chloroform. The procedure was continued as described under the preparation of calibration graphs [24]. BCG method : Ten tablets were finely powdered and mixed .An accurately weighed quantity equivalent to the drug base concentration, mentioned in the standard solution p reparation, was dissolved in 200 ml of methanol by using volumetric flask .The procedure was continued as described under the preparation of calibration graghs [24]. Results and Discussion Charge transfer complexation: The reaction of amitriptyline with TCNE in acetonitrile solvent results in the formation an intense red brown color complex, which exhibits an absorp tion maximum at 470 nm (Fig 1). This absorption band formed is the results of the formation of charge-transfer complex through the interaction of TCNE as a π - acceptor and the studied drug as n-donor followed by the formation of colored radical anion accord ing to the following scheme [22,24,35,36]: IBN AL- HAITHAM J. FO R PURE & APPL. SCI. VO L. 22 (4) 2009 Formation of a radical anion in such molecular interactions was confirmed by electron-spin resonance measurements [37]. Optimization of experimental variables: i. Univariate method Effect of pH: The effect of pH on the development of the colored complex between amitriptyline and TCNE is shown in (Fig 2). The pH being adjusted with few drops of 0.1 M HCl and 0.1 M NaOH. M aximum and constant absorbance were obtained in the pH range 9.0-9.5.The absorbance decreased at pH value above 9.5 and below 9.0. Hence a pH of 9.0 was used in all the subsequent experimental work. Effect of reagent: Various vo lumes of TCNE solution were added to 40µg.ml -1 of amitriptyline solution. 1.5 ml of 1.6x 10 -4 M of TCNE was found to be enou gh to develop the color to its full intensity and was considered to be the op timum for the concentration range of amitrip tyline 6.0-75 µg.ml -1 (Fig 3). Effect of reaction time: The color intensity reached a maximum after the amitriptyline was reacted with TCNE for 30 minutes (Fig 4), therefore 30 minutes development time was selected as op timum in the general procedure . The color obtained was stable for at least 2 hours. S toichiometry of the complex : The stoichiometry of the reaction between amitriptyline and TCNE was studied by mole ratio method (Fig 5). The results obtained shows that 1:1 amitriptyline to TCNE was formed at 470nm, therefore the formation of the complex can be represented as in following scheme [22,35] : IBN AL- HAITHAM J. FO R PURE & APPL. SCI. VO L. 22 (4) 2009 ii. Simplex optimization: To set the simplex op timization of the three studied variables, four experimental conditions should be chosen involve values for pH, reagent volume and standing time. The values of the four experimental conditions were selected with specific boundaries for each at which it affects the absorpt ion signal of the colored product (table1). The absorbencies of these four initial experiments were measured and the results were feed to the computer program. The program then starts simplex by searching the worse absorption signal and reflects it in ahyper-plane of the remaining points to p roduce a new set of experimental conditions, which were applied to carry out the experiment and the measured absorption signal was feeded again to the program. The process is repeated successively until optimum conditions were obtained (i.e. conditions yielding highest absorption signal). The procedure is continued for further few experiments to ensure that the optimum conditions are reached (Table 2 and Figure 6). Values of the results obtained by applying simplex program are shown in Figure 7. Calibration graph: Employing the experimental conditions described under procedure, a linear calibration graph for amitriptyline is obtained (Fig 8), which shows that Beers law was obeyed in the concentration range 6.0-70 µg.ml -1 . Ion-pair complexation: The amitriptyline solution reacted with BCG solution in aqueous solution in acidic medium to form a yellow color ion pair complex, which exhibits an absorption maximum band at 415nm against reagent blank (Fig 9). Optimization of experimental variables: IBN AL- HAITHAM J. FO R PURE & APPL. SCI. VO L. 22 (4) 2009 i. Univariate method: Effect of pH: In order to established the optimum pH range , amitriptyline solution was mixed with aspecified volume of BCG , and then the pH was adjusted to a value between 2.0-6.0 with a few drops of 0.1N NaOH or 0.1N HCl .M aximum and constant absorbances were obtained in the pH range 3.5-4.0 (Fig 10) . The absorbance was decreased at pH value above 4.0 and below 3.50. Hence a pH of 4.0 was used in all the subsequent experimental work. Effect of reagent: The influence of excess reagent concentration on the absorbance of the complex is illustrated in (Fig 11). One ml of 1.6x10 -4 M solution of BCG was found enough to develop the color and reached its maximum intensity . Effect of shaking time: The optimum shaking time for the complete extraction of the ion pair complex with chloroform was studied from 30 second to 3 minutes (Fig12). It was found that the minimum shaking time for complete extraction was 2 minutes at room temperature (25±1 C o ) . S toichiometry of the complex: The stoichiometry of the reaction between amitriptyline and BCG was studied by mole ratio method (Fig13). The results obtained shows that 1:1 amitriptyline to BCG was formed at 415 nm, therefore the formation of the complex can be represented as in following scheme [24,25] : IBN AL- HAITHAM J. FO R PURE & APPL. SCI. VO L. 22 (4) 2009 ii. Simplex optimization: The same steps of simplex program, within the given boundary conditions (Table 3), were followed to optimize the experimental conditions. Results are shown in Figure 14, 15 and Table 4. Calibration graph Employing the experimental conditions, a linear calibration graph for amitriptyline is obtained (Fig 16), which shows that Beers law was obeyed in the concentration range of 8.0-100 µg.ml-1. S pectral characteristic of the two proposed methods: Under the optimum experimental cond itions of the two proposed methods, the regression plots showed that there were linear dependence of absorbance signals on the concentration of the drug in the ranges given in Table 5. The regression equations and correlation coefficients, which were obtained by the linear least –squares treatment of the results in addition to the molar absorptivites, detection limits , and Sandell sensitivities are given in Table 5. The accuracies of the two proposed methods were established by performing seven replicate analyses on standard solutions containing three different amounts of drug and calculating the percentage error. The precisions were determined by calculating the relative standard deviations (RSD) for seven determinations at each level Table 6. It is clear from the results that at all of the three studied levels , the values of the mean ( ) were less than the values of indeterminate error ( ), indicating that no significant differences existed between the mean and the true values. Table 7 shows that the two proposed methods have acceptable linearity ranges, acceptable precisions and accuracies when they compared with other methods. Analytical application: The proposed methods were applied to determine amitriptyline in pharmaceutical preparation tablets. The results, presented in Table 8, reveal that the recover ies were in the range of 94.56 to 100.20 %, reflecting high accuracies and precisions of the proposed methods as indicated by low RSD values. References 1- Biederman,J. and Spencer ,T. (1999). Biol Psychiatry , 46(9) :1234-42. 2- Mico,J.; Ardid,D. ; Berrocoso,E. and Eschalier, A. (2006) Trends Pharmacol Sci , 27(7 ): 348-54. 3- Kou,H.S. ; Chen,C.C. ; Huang,H.Y. ; Ko,W.K. ; Wu,H.I. and Wu S.M. (2004) Analytica Chimica Acta, 525(1): 23-30. 4- Wu,S.M . and Chen,S.H. (2000).Analytica Chimica Acta, 443(1-2) :125-129. 5- Yoshida ,H. ; Hidaka ,K. ; Ishida ,J. ; Nohta ,H. and Yamaguchi , M . (2000).Analytica Chimica Acta, 413(1)18 M ay : 137-145(9). 6- Linden,R.; Antunes, M .V .; Ziulkoski,A.L.; Wingert, M .; Tonello,P. ;Tzvetkov M . and A.A.Souto.(2008). J.Braz.Chem.Soc. 19(1): 34-41. 7- Ulrich,S. ; Isensee,T. and Pester U. (1996). 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Analytical letters, 35(7):1163- 1170. 17- Karp inska,J. and Suszynska ,J. (2001). Journal of Trace and M icroprobe Techniques, 9(3): 355-364. 18- Onah ,J.O. (2005). Global Journal of Pure and Applied Sciences, 11(2) : 237-240. 19- Gamal,A. ; Hussan,F. ; Ibrahim,A. and Abdel-Nasser A. (2003). Analytical sciences, 19 : 281-287. 20- Salem ,H. (2008) .African Journal of Pharmacy and Pharmacology, 2 (7):136-144. 21- Basavaiah ,K. and Charan ,V.S (2002). Science Asia 28 : 359-364. 22- Walash,M .; Sharaf-EL-Din, M .; M etawalli ,E.S and Redashabana ,M. (2004).Arch Pharm Res, 27(7): 720-727. 23- Betello ,J.C. and Perez, G. (1995). Talanta, 42(1): 105-108. 24- Elham, A.; Suzan,M . ; Hisham ,E. and M agda, M . (2002). Microchim .Acta, 140: 175-182. 25- Basavaiah ,K. and Charan, V.S. (2004). Science Asia, 30 : 163-170. 26- Spendely,W. ; Hext ,G.R. and Himsworth F.R. (1962). Technometrics, 4 : 441-461. 27- Nelder ,J.A. and M ead ,R.A (1965). Computer Journal, 7 : 308-313. 28- Alberg ,E.R. and Gustavsson A.G. (1982).Analytica Chimica Acta, 144: 39-53. 29- Sultan,S.M .; Suliman, F.E. ; Duffuaa S.O. and Abu-Abdoun ,I.I. (1992).Analyst, (7): 1179-1186. 30- Sultan ,S.M . and El-M ubarak ,A.H. (1996). Optimized by the modified simplex procedure, Talanta, 43 : 569-576. 31- Momenbeik,F. ; M omeniz ,Z. and Kharasani ,J.H. (2005). Journal of Pharmaceutical and Biomedical Analysis, 37(2) : 383-387. 32- Pulgarin,J.A.; Molina ,A. and Pardo ,M.I. (2002). Talanta, 57 : 795-805. 33- Wade,A.P. University college Swansea, Wales, cited in S.Al-Najafi.(1984) .Thesis, university of Wales, university college of Swansea 34- Vogel,I. Atext books of macro and semimicro quant itative in organic analysis (1959).4 th .Edn.page :645. 35- Abdellatef ,H.E. (1998). Journal of Pharmaceutical and B iomedical Analysis, 17: 1267-1271. 36- Al-Ghannam ,S. and Belal ,F. (2002) J.AOAC Int , 85 : 1003. 37- Abdel Hamid, M.E.; Abdel Salam,M .;M ahrous, M .S. and Abdel-khalek, M.M . (1985). Talanta, 36 :1002-1004. IBN AL- HAITHAM J. FO R PURE & APPL. SCI. VO L. 22 (4) 2009 Fig.(1): Absorption spectra of: (A) 40 µg.ml -1 amitriptyline, 2.4 x 10 -4 M TCNE at pH=9.0 against reagent blank; (B) 2.4 x 10-4M TCNE in acetonitrile against distilled water. Fig.(2): Effect of pH on the absorbance of : 40 µg.ml-1 amitriptyline, 2.4 x 10-4 M TCNE at 470 nm Fig.(3): Effect of reagent volume on the absorbance of 40 µg.ml -1 amitriptyline solution. IBN AL- HAITHAM J. FO R PURE & APPL. SCI. VO L. 22 (4) 2009 Fig.(4): Effect of standing time on the absorbance of 40 µg.ml -1 amitriptyline, 2.4 x 10 -4 M TCNE at pH=9.0. Fig.(5): Mole ratio plot of amitriptyline determined via charge-transfer method. Fig.(6): Experimental simplex study of variables resulting in pH=9, reagent volume=1.5ml, and standing time=30min. IBN AL- HAITHAM J. FO R PURE & APPL. SCI. VO L. 22 (4) 2009 Fig. (7): Optimization of absorption signal via simplex changing of pH, reagent volume, and standing time. Fig.(8): Calibration graph of amitriptyline with 2.4 x 10 -4 M TCNE at 460 nm. Fig.(9): Absorption spectra of: (A) 45 µg.ml -1 amitriptyline, 1.0 x 10 -4 M BCG at pH=3.5 against reagent blank; (B) 1.0 x 10 -4 M BCG in chloform against distilled water. IBN AL- HAITHAM J. FO R PURE & APPL. SCI. VO L. 22 (4) 2009 Fig.(10): Effect of pH on the absorbance of : 45 µg.ml -1 amitriptyline, 1.0 x 10 -4 M BCG at 415 nm Fig.(11): Effect of reagent volume on the absorbance of 45 µg.ml -1 amitriptyline solution. Fig.(12): Effect of shaking time on the absorbance of 45 µg.ml -1 amitriptyline, 1.0 x 10 -4 M BCG. IBN AL- HAITHAM J. FO R PURE & APPL. SCI. VO L. 22 (4) 2009 Fig.(13): Mole ratio plot of amitriptyline determined via ion-pair method Fig.(14): Experimental simplex study of variables resulting in pH=1.2, reagent volume=1.2ml, and shaking time=90 sec. Fig.(15): Optimization of absorption signal via simplex changing of pH, reagent volume, and shaking time. IBN AL- HAITHAM J. FO R PURE & APPL. SCI. VO L. 22 (4) 2009 Fig.(16): Calibration graph of amitriptyline with 1.0 x 10 -4 M BCG at 415 nm. Table (1): Boundary conditions for the studied variables Variable range pH 6-11 Reagent volume (ml) 0.3-3.0 Standing time (min) 5-60 Table( 2): Absorbance for each of the simplexes in the optimization of color producing reaction variables Table (3): Boundary conditions for the studied variables Variable range pH 2-6 Reagent volume (ml) 0.2-2.0 Standing time (sec) 30-180 IBN AL- HAITHAM J. FO R PURE & APPL. SCI. VO L. 22 (4) 2009 Operation pH Reagent Volume(ml ) Time (min) Absorbance Simplex 1 8.0 1.2 35 0.200 Simplex 2 10.0 2.4 20 0.267 Simplex 3 8.5 2.1 15 0.239 Simplex 4 6.0 1.8 30 0.246 Simplex 5 9.0 3.0 5 0.212 Simplex 6 8.5 1.8 30 0.297 Simplex 7 8.0 1.8 35 0.287 Simplex 8 11.0 2.1 30 0.279 Simplex 9 8.0 1.2 35 0.279 Simplex 10 10.0 2.4 25 0.259 Simplex 11 8.5 1.5 35 0.310 Simplex 12 8.5 1.8 30 0.297 Simplex 13 6.0 1.2 35 0.246 Simplex 14 9.5 1.8 30 0.335 Simplex 15 10.0 1.8 25 0.289 Simplex 16 9.5 1.8 30 0.335 Simplex 17 10.0 1.8 30 0.311 Simplex 18 10.5 2.1 25 0.259 Simplex 19 9.0 1.5 30 0.350 Simplex 20 9.5 1.8 30 0.335 Simplex 21 8.5 1.5 30 0.310 Simplex 22 9.5 1.8 30 0.335 Table (4): Absorbance for each of the simplexes in the optimization of color producing reaction variables Operation pH Reagent Volume(ml) Time (sec) Absorbance Simplex 1 3.0 1.6 90 0.203 Simplex 2 3.0 0.6 60 0.166 Simplex 3 3.5 1.4 120 0.240 Simplex 4 5.5 1.8 30 0.138 Simplex 5 2.0 0.8 120 0.180 Simplex 6 3.0 2.0 120 0.198 Simplex 7 4.5 2.0 120 0.198 Simplex 8 2.0 1.2 120 0.200 Simplex 9 3.0 0.8 120 0.207 Simplex 10 4.5 1.4 120 0.221 Simplex 11 4.0 1.0 120 0.250 Simplex 12 5.0 1.6 120 0.212 Simplex 13 4.5 0.6 120 0.193 Simplex 14 3.5 1.2 90 0.250 Simplex 15 3.0 0.8 120 0.207 Simplex 16 4.0 1.2 120 0.250 Table(5): Analytical characteristics for the two methods. Parameter TCNE method BCG method linear dynamic range (µg.ml -1 ) 6.0-70 8.0-100 Regression equation Abs=0.008Conc.+0.027 Abs=0.005Conc.+0.007 Slope (b) (l.mg -1 .cm -1 ) 0.008 0.005 Intercept (a) 0.027 0.007 Correlation coeff icient 0.9998 0.9999 M olar absorptivity l.mol -1 .cm -1 2275 1475.50 Detection limit (µg.ml -1 )* 0.043 0.034 Sandell sensitivity (µg.ml-1) 0.122 0.188 *Calculated for single analysis at 99.9 confidence limit. IBN AL- HAITHAM J. FO R PURE & APPL. SCI. VO L. 22 (4) 2009 Table(6): Evaluation of accuracy and precision of the two methods. Item TCNE method BCG method Concentration of amitriptyline taken ( µg.ml- 1 ) 15.00 25.00 35.00 30.00 20.00 50.00 Concentration of amitriptyline found ( µg.ml -1 ) * 15.03 24.85 34.75 30.13 20.24 50.21 Error % 0.20 0.60 0.71 0.43 0.24 0.42 Standard deviation 0.11 0.59 0.59 0.39 0.41 0.30 R.S.D % (n= 7) 0.76 2.40 1.71 1.29 2.06 0.61 0.03 0.15 0.25 0.13 0.24 0.21 0.10 0.52 0.52 0.34 0.37 0.27 * M ean value of seven determinations (N) at each level. =mean value , µ= true value. t= 2.36 for n=7 at 95% confidence level. s =standard deviation. Table (7): Comparison of linearties of the two proposed methods with those from other spectrophotometric methods for determination of amitriptyline in pharmaceutical formulation. * Extractive procedure Reagent Beers law limit µg.ml-1 References Niobium (V) thiocyanate 1.0-12 16 - 1.0-30 12 Potassium thiocyanate * 3.0-60 14 Ammonium molybdate 1.0-140 15 Bromocresol purple * 30-200 13 Chloranilic acid 8.7-90 18 TCNE 6.0-75 This work BCG 8.0-100 This work IBN AL- HAITHAM J. FO R PURE & APPL. SCI. VO L. 22 (4) 2009 Table (8): Results of analyses of pharmaceutical preparations containing amitriptyline by TCNE method and BCG method. § n = 4 * M arked by S.D.I, Iraq. ♦ M arked by MSD-USA. ◊ M arked by HM -Holden BV-Holland Depresol * 25mg Tryp tizol ♦ 25mg Amitriptyline ◊ 25 mg M ethod Taken (µ.ml - 1) Found (µ.ml - 1) § M ean Recovery% § R.S.D.% Taken (µ.ml - 1) Found (µ.ml - 1) § M ean Recovery% § R.S.D.% Taken (µ.ml - 1) Found (µ.ml - 1) § Mean Recovery% § R.S.D.% 15.00 14.95 99.68 0.98 15.00 14.84 98.94 1.98 25.00 24.84 99.38 1.18 25.00 24.84 99.38 1.18 30.00 29.42 98.09 0.90 35.00 34.84 94.56 0.81 Charge- transfer method 45.00 44.52 98.94 0.71 45.00 44.52 98.94 0.71 50.00 49.77 99.55 1.84 15.00 14.92 99.46 0.82 15.00 14.91 99.40 0.71 20.00 19.74 98.71 0.66 30.00 29.87 99.56 0.66 35.00 35.07 100.20 0.50 40.00 39.89 99.75 0.91 Ion-pair method 60.00 59.71 99.51 0.87 60.00 59.60 99.33 1.01 70.00 69.92 99.88 0.41 37-M .E.Abdel Hamid, M . Abdel Salam, M.S. M ahrous and M .M.Abdel-Khalek, Utility of 7,7,8,8-tetracyanoquinodimethane and p- chloranilic acid in the qualitative and quantitative analysis